The following polypeptide was digested with the proteolytic enzyme trypsin. NH2-

Question

The following polypeptide was digested with the proteolytic enzyme trypsin. NH2-Met-Gln-Tyr-Thr-Ser-Gly-Asn-Arg-Trp-Ala-Gly-Val-Leu-Cys-Pro-Lys-Gln-Glu-Asp-Ser-Asp-Glu-Tyr-Thr-COOH

B. You are asked to separate the peptides from one another by a two-step column chromatography procedure. At your disposal are ion exchange columns (cation or anion, based on your choosing), and a hydrophobic column. Describe how you would carry out chromatography to accomplish this separation. Your description should explain how the various fragments separate from each other in each step

Explanation / Answer

Trypsin digestion of this polypeptide will yield

NH2-Met-Gln-Tyr-Thr-Ser-Gly-Asn-Arg- COOH (P-P-P-P-P-H-P-C; +1 charge) fragment 1

NH2-Trp-Ala-Gly-Val-Leu-Cys-Pro-Lys- COOH (P-H-H-H-H-P-H-C; +1 charge) fragment 2

NH2-Gln-Glu-Asp-Ser-Asp-Glu-Tyr-Thr-COOH (P-C-C-P-C-C-P-P; -4 charge) fragment 3

P : Polar residue

H : Hydrophobic residue

C : Charged residue

The best way to load these fragments first on anion-exchange column which will retain only fragment 3 (because of its high negative charge) and other two fragments will be in wash. Now load the wash having fragments 1 and 2 on hydrphobic column which will retain fragment 2 (due to high hydrophobicity). Fragment 1 will be in wash.

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